CN103641913B - A kind of Hansenula yeast HCP preparation method for antibody, antibody and its application is made - Google Patents
A kind of Hansenula yeast HCP preparation method for antibody, antibody and its application is made Download PDFInfo
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Abstract
The invention discloses a kind of anti-Hansenula yeast host protein (HCP) antibody, the anti-Hansenula yeast host protein antibody is polyclonal antibody, is made again after antibody purification through animal immune after Hansenula yeast host protein is made with Hansenula yeast bacterial strain.Present invention also offers the preparation method of the antibody, this method includes preparing Hansenula yeast host cell holoprotein, by adding antigen purification step in the method for routine immunization animal, so that animal to Hansenula yeast whole host cell after immune response is produced, strengthen the immunoreactivity for part weak immunogenicity host protein, exempt from reinforced host's residual protein antibody so as to be made low；Test result indicates that, antibody provided by the invention can be applied in ELISA detection host's residual proteins, sensitivity can not only be improved, and it is all general by host's residual protein detection of the biological products of expressed by Hansenula yeast for major part, therefore there is very wide application prospect.
Description
Technical field
The invention belongs to biological medicine and detection technique field, specifically, is related to a kind of preparation side of detection antibody
Method, more specifically to a kind of preparation side of Hansenula yeast HCP (Host cell protein, HCP, host protein) antibody
Method, this method prepare gained antibody, and application of the antibody in correlation detection technology.
Background technology
The characteristics of saccharomycete is both fast with prokaryotes growth as unicellular eukaryote, and genetic manipulation is simple, has again
There are the gene expression regulation similar to higher eucaryote and posttranslational modification mechanism, therefore be that production has eucaryote activity
The preferable expression system of albumen.Wherein multiple-shaped nuohan inferior yeast (Hansenula polymorpha) is a kind of reason generally acknowledged in the world
The heterologous gene expression system thought is existing in recent years big better than Escherichia coli and the heterologous gene expression system of other saccharomycete
Amount be difficult to the foreign protein of high efficient expression in expressed by Hansenula yeast system all successful expression；It is in biological products industrialization
Application value is as follows：1) for exogenous origin gene integrator on chromosome, recombinant bacterial strain genetic stability is high；2) frequency of non-homogeneous restructuring
Height, easily obtain the transformant that high copy is integrated；3) efficiently start using strong promoters such as methanol oxidase (MOX) promoters
The expression of foreign gene；4) expression of foreign protein is carried out in peroxidase body, can protect it from intracellular protease
Degraded, and reduce the toxic action to cell；5) high temperature resistant, optimum growth temperature are 37-43 DEG C, and growth rate is fast, big rule
The incubation time of mould fermentation is short.Currently employed multiple-shaped nuohan inferior yeast has restructuring B-mode as the biological products that expression system has listed
Hepatitis virus vaccine, hirudin etc.；There is the report of a variety of foreign proteins successful expression in multiple-shaped nuohan inferior yeast in addition.
Contain substantial amounts of host protein (Host cell protein, HCP), DNA and endotoxin etc. in host cell,
It may be polluted to biological products.Wherein the potential immunogenicity of host protein is possible to induce body to produce corresponding antibodies,
Generation immune response；And its potential " adjuvant effect " may also can cause body to produce antibody to medicine, and then influence medicine
The effect of thing.High sensitivity, reproducible host protein detection method are not only to ensure that biological products are safely and effectively crucial,
And the important parameter of production process control and process optimization.
At present, yeast host method of protein detection mainly has ELISA (ELISA), polyacrylamide gel electrophoresis
(SDS-PAGE), Capillary Electrophoresis (CE), reversed-phase HPLC, chromatography of ions etc..《Chinese Pharmacopoeia》Host's egg in three (2010 editions)
White residues detection method uses ELISA (ELISA).Anti- host protein antibody used leads in ELISA detection method
It is often that after host cell is crushed, host protein is obtained by preliminary treatment, is obtained after immune animal.Host cell breaking method
Including：Organic reagent cracks；The enzymatic reaction of the enzymes such as lysozyme；The physics such as ultrasound, high-pressure homogenization crush.Preliminary manner
Including：Separation of solid and liquid；Be concentrated by ultrafiltration etc..
Conventional yeast HCP preparation method for antibody, because HCP molecular size ranges differ, complicated component, exempt between each composition
Epidemic focus sex differernce is big, and is not readily available suitable HCP antibody.Because yeast HCP whole compositions are immunized conventional method
After animal, the HCP compositions that antibody is big mainly in combination with molecular weight and immunogenicity is strong are obtained, for low molecule amount and immunogenicity
Weak HCP compositions are without binding ability.Using antibody caused by this preparation method, can cause by ELISA (enzyme linked immunologicals
Method) when HCP is remained in detection biological products sensitivity substantially reduce, and because the HCP compositions of part low immunogenicity can not
Detect and cause actual measured value relatively low.
Summary, a kind of suitable Hansenula yeast for being used for Hansenula yeast host protein residues detection is there is no at present
HCP antibody and the method detected using the antibody, the more matched reagent and kit without correlation.
The content of the invention
For existing detection technique above shortcomings, in order to residual host's egg in Hansenula yeast fermenting organism product
White detection provides a kind of detection method that is fast and effective, simple and reliable, being applicable large-scale promotion application, and the present invention is by existing
The method that Hansenula yeast host protein prepares immune antiboidy is improved, and preparation-obtained antibody has that immunogenicity is strong, inspection
The characteristics of surveying high sensitivity.
To achieve these goals, the technical solution adopted in the present invention is：One kind is new to prepare Hansenula yeast HCP antibody
Method, pass through in the method for routine immunization animal add antigen purification step so that animal is to Hansenula yeast whole place
After chief cell produces immune response, strengthen the immunoreactivity for part weak immunogenicity host protein, it is low so as to be made
Exempt from reinforced HCP antibody；
As a kind of preferred scheme, the new method for preparing Hansenula yeast HCP antibody, comprise the following steps that：
A) 2~4 kinds of conventional Hansenula yeast strains are taken to carry out seed culture and induced expression respectively, induced expression 48~90 is small
When after, collect thalline；
B) by the thalline of above-mentioned induced expression respectively, equal weight is respectively taken to be mixed, clasmatosis, separation of solid and liquid,
Bacteria break supernatant is collected, filtration sterilization, mixing Hansenula yeast HCP holoproteins are made；
C) suitable adjuvant immunity animal is added using mixing Hansenula yeast HCP holoproteins, be immunized three times altogether, obtained middle
Antibody；
D) Antibody preparation affinity chromatography glue among using, affinity purification Hansenula yeast HCP holoproteins, obtains Hansenula yeast
HCP is low to exempt from albumen；
E) continue immune animal on the basis of step c) using the low albumen of exempting from of Hansenula yeast HCP, altogether three times, it is inferior to obtain the Chinese
Yeast HCP is low to exempt from reinforced antibody；
Wherein, 2~4 kinds of conventional Hansenula yeast strains described in step a) are wild type, preferably 3 kinds, are respectively
ATCC14754, ATCC34438 and ATCC66057；
Immune animal described in step c), altogether three times, respectively carried out at the 0th, 14,28 day；
Antibody purification Hansenula yeast HCP holoproteins obtain HCP low the step of exempting from albumen among use described in step d)
For Hansenula yeast HCP holoproteins is appropriate, among addition in antibody affinity chromatography glue, collection flows through protein sample, and the as Chinese is inferior
Yeast host cell is low to exempt from albumen, immune for follow-up fourth, fifth, six time；
Immune Animal Procedures described in step e), using the low albumen of exempting from of Hansenula yeast made from step c) as antigen and adjuvant
Animal is immunized three times altogether, was respectively carried out at the 42nd, 49,56 day；The animal can be mouse, rat, cavy, rabbit, sheep, horse,
Preferably rabbit and cavy；
The adjuvant is selected from Freund's adjuvant, aluminum hydroxide adjuvant, Aluminium phosphate adjuvant, Liposome Adjuvant, CpG adjuvants, preferably
For Freund's adjuvant, most preferably Freund's complete adjuvant or incomplete Freund's adjuvant；
Exempt from reinforced antibody, the Hansenula yeast HCP it is a further object to provide a kind of Hansenula yeast HCP is low
Low to exempt from reinforced antibody be polyclonal antibody, is passed through again through animal immune after Hansenula yeast host protein is made with Hansenula yeast bacterial strain
It is made after antibody purification；
As a kind of preferred scheme, also include antigen purification step in the animal immune so that immune animal is right
After whole host cells produce immune response, strengthen the immunoreactivity for weak immunogenicity host protein；
As still more preferably scheme, the antigen purification step is using animal immune moderate resistance Hansenula yeast host's egg
Bai Kangti middle antibody purification Hansenula yeast host cell holoprotein, obtains that Hansenula yeast host cell is low exempts from albumen；
Another purpose of the present invention is to provide the low applications for exempting from reinforced antibody of Hansenula yeast HCP, primarily as work
Property composition be used for the reagent or kit that prepare biological products host protein residual quantity prepared by detection expressed by Hansenula yeast system；
As a kind of preferred scheme, the product of the detection Hansenula yeast host protein is detection reagent or detection kit；
Alleged Hansenula yeast host protein, as Hansenula yeast HCP in the present invention, and Hansenula yeast host's holoprotein,
Or Hansenula yeast holoprotein, refer to cultured Hansenula yeast thalline through collecting, it is broken, centrifuge, collect supernatant,
Obtained product after filtration sterilization, can directly choose doses, for animal to be immunized；
Alleged Hansenula yeast host is low in the present invention exempts from albumen, refer in Hansenula yeast holoprotein part molecular weight compared with
Small, immunogenicity is relatively low, is blended in Hansenula yeast holoprotein and the composition that animal is not readily available antibody is immunized with conventional method；
Compared with prior art, the present invention carries for the deficiency of Hansenula yeast host protein detection means in the prior art
High anti-Hansenula yeast host protein antibody of a kind of detection sensitivity and preparation method thereof has been supplied, antigen is added in immune animal
Purification step so that animal is strengthened for weak immunogenicity host protein after immune response is produced to whole host cells
Immunoreactivity.Test result indicates that antibody provided by the invention can be applied in ELISA detections HCP, can not only improve sensitive
Degree, and because the present invention is when preparing yeast host holoprotein, employ conventional several wild type Hansenula yeast bacterial strains by
Certain proportion is mixed, widely applicable for most of all general by the biological products HCP detections of expressed by Hansenula yeast.
Brief description of the drawings
Fig. 1 is the present invention and prior art standard curve control figure.
Embodiment
The present invention is made further in detail, intactly to illustrate with reference to embodiment and comparative example.In the following example not
The experimental method of actual conditions is indicated, generally according to normal condition, such as Smabrook et al., molecular cloning：Laboratory manual
(New York：Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to manufactory
Condition proposed by business.Ratio and percentage are based on weight, unless stated otherwise.
Used animal, instrument and equipment, reagent and its preparation etc. are from commercially available in the experiment of following examples.
Polymorpha strain：ATCC14754, ATCC34438, ATCC66057
YPD culture mediums：Autogamy, 1% dusty yeast, 2% peptone, 2% glucose.
BMMY culture mediums：Autogamy, 1% dusty yeast, 2% peptone, 100mM kaliumphosphate buffers (pH6.0), 4 × 10-5%
Biotin (biotin, Sigma companies), 1.34%YNB (yeast nitrogen alkali, BD companies), use 3% methanol of preceding addition.
High pressure homogenizer：ATS AH-100B
High speed freezing centrifuge：Thermo Fisher lynx4000
Freund's complete adjuvant：Sigma companies
Incomplete Freund's adjuvant：Sigma companies
Sephadex G25 chromatographic columns：GE companies
Affinity column UNOSPHERE：Bio-rad companies
Other biological reagents and chemical reagent used in the present invention, it is to analyze pure or analysis unless otherwise instructed
Pure above rank.
The preparation of the Hansenula yeast HCP antibody of embodiment 1
1. the preparation of Hansenula yeast host's holoprotein
Using the polymorpha strain from American Type Culture Collection center (ATCC), it is more wide to choose application
Three plants of general wild type reference cultures, numbering ATCC14754, ATCC34438, ATCC66057.Above-mentioned three plants of bacterial strains are distinguished
YPD culture mediums are seeded to, 37 DEG C of cultures, change BMMY culture medium inducible gene expressions, addition 0.5% in every 24 hours after 24 hours into
Methanol, after expressing 72 hours, 10000rpm centrifugations 10min collects thalline.The simulation completely of above-mentioned thalline culture Induction Process contains
Culture inductive condition during the Hansenula yeast engineered strain production of foreign gene.
By it is above-mentioned be made three plants of bacterial strains thalline respectively take 50g to mix, add 4 DEG C of precoolings cushioning liquid A (50mM PB,
0.2M NaCl, 0.5%Tween-80, pH7.0) 600mL, mixed in stirring on magnetic stirring apparatus to complete.Using high-pressure homogeneous
Machine (ATS AH-100B) high pressure crushes above thalline suspension, adjusts homogenizing valve, it is 1200bar to control brokenly bacterium pressure.Repeat with
Upper high pressure shattering process 3-4 times.High speed freezing centrifuge (Thermo Fisher lynx4000) centrifugal clarification breaks bacterium solution, setting
Condition 10000rpm, 30min, 8 DEG C, centrifuge, collect the supernatant after centrifugation.Supernatant is diluted to cushioning liquid A
Protein concentration 500ug/mL, packing after -80 DEG C preservation degerming with 0.22um membrane filtrations；It is complete that Hansenula yeast host cell is made
Albumen.
2. animal immune program
Take three rabbits and two cavys to carry out animal immune, carry out six times respectively and be immunized, it is isometric with adjuvant using antigen
Mixing, emulsification use syringe mixing method：The Freund's adjuvant of equivalent and antigenic solution are sucked in two syringes respectively, two notes
It is connected between emitter with a thin sebific duct, pays attention to emptying air, then alternately promotes needle tubing, untill sticky emulsion is formed,
As emulsification is abundant.After emulsification fully, animal is immunized.Just exempt to use Freund's complete adjuvant, then immune all uses Freund not
Freund's complete adjuvant.Immunizing dose is 0.5mg/ rabbits, 0.25mg/ cavys, using dorsal sc multi-point injection.
Specifically immune programme for children is：Just exempt from (0 day, Freund's complete adjuvant), antigen is Hansenula yeast host cell holoprotein；Two
Exempt from (14 days, incomplete Freund's adjuvant), antigen is Hansenula yeast host cell holoprotein；Three exempt from that (28 days, Freund was not exclusively helped
Agent), antigen is Hansenula yeast host cell holoprotein.
After three exempt from, a rabbit is put to death, blood sampling, obtain middle antibody；It is thin with middle antibody purification Hansenula yeast host
Born of the same parents' holoprotein, obtains that Hansenula yeast host cell is low exempts from albumen, and wherein Hansenula yeast host cell is low exempts from the albumen specifically side of preparation
Method is：After being immunized for the third time, a rabbit is put to death, 20mL rabbit anteserums is taken, adds one times of normal saline dilution, be added dropwise to
(NH4)2SO4Saturated solution after being sufficiently mixed, stands 5-6 hours, 12000rpm centrifugation 20min, abandoned to final concentration of 50%
Clearly.Precipitation 20mL physiological saline solution, is added dropwise to (NH4)2SO4Saturated solution is to final concentration of 33%, after being sufficiently mixed,
5-6 hours are stood, 12000rpm centrifugation 20min, abandon supernatant；Precipitation uses 10mL physiological saline solutions, and the supernatant of centrifugation passes through
Sephadex G25 chromatographic columns (GE companies) desalination, changes liquid (pH7.4) into PBS solution.Cross affinity column UNOSPHERE
(Bio-rad companies), rabbit igg antibody is obtained with 0.1M pH3.0 citrate buffer solution elution；The antibody is inferior mainly in combination with the Chinese
The high antigen protein of macromolecule, immunogenicity in yeast host cell holoprotein.Use Affi-Gel Hz
Immunoaffinity kit (Bio-rad) kit, gained rabbit igg antibody is changed into liquid to Coupling Buffer, used
NalO4. oxidised antibody, liquid Coupling Buffer being changed again, and being coupled to Affi-Gel Hz Gel, rabbit igg antibody is made
Affi-Gel.Use above-mentioned rabbit igg antibody Affi-Gel, purification step 1) made from Hansenula yeast host cell holoprotein；By the Chinese
Inferior yeast host cell holoprotein sample 10mL, adds rabbit igg antibody affinity chromatography (Affi-Gel 20mL), and collection flows through albumen
Sample, as Hansenula yeast host cell is low exempts from albumen, immune for follow-up fourth, fifth, six time.
Remaining two rabbits and two cavys carry out subsequently being immunized three times again；Four exempt from (42 days, incomplete Freund's adjuvant),
Antigen exempts from albumen for Hansenula yeast host cell is low；Five exempt from (49 days, incomplete Freund's adjuvant), and antigen is that Hansenula yeast host is thin
Born of the same parents are low to exempt from albumen；Six exempt from (56 days, incomplete Freund's adjuvant), and antigen exempts from albumen for Hansenula yeast host cell is low.Put to death within 63 days,
Blood sampling, serum purifying, concretely comprises the following steps serum adding one times of normal saline dilution, be added dropwise to (NH4)2SO4Saturated solution is dense to end
Spend for 50%, after being sufficiently mixed, stand 5-6 hours, 12000rpm centrifugation 20min, abandon supernatant.Precipitation 20mL physiological saline
Dissolving, is added dropwise to (NH4)2SO4Saturated solution after being sufficiently mixed, stands 5-6 hours, 12000rpm centrifugations to final concentration of 33%
20min, abandon supernatant；Precipitation 10mL physiological saline solutions, (GE is public by Sephadex G25 chromatographic columns for the supernatant of centrifugation
Department) desalination, change liquid (pH7.4) into PBS solution.Affinity column UNOSPHERE (Bio-rad companies) is crossed, uses 0.1M
PH3.0 citrate buffer solution elution obtains.After respectively obtain rabbit and the anti-Hansenula yeast host protein polyclonal antibody of cavy.
The anti-Hansenula yeast host protein antibody titer measure of embodiment 2
Using ELISA method detection antibody titer, it is specially：Using coating buffer (0.05M NaHCO3, pH9.6) and by the inferior ferment of the Chinese
Female host cell holoprotein is diluted to 10ug/mL, and 100ul/ holes, 4 DEG C of coatings are overnight.Coating buffer is removed, two are washed with PBS solution
It is secondary.0.3mL confining liquids (5% skimmed milk power+PBS) are added per hole and are incubated 2 hours in 37 DEG C；Coating buffer is removed again, and PBS solution is washed
Wash twice.The anti-Hansenula yeast host protein antibody of rabbit-anti/cavy that above-mentioned purifying is obtained is diluted to 1mg/mL, then series respectively
10 times of dilutions, dilution buffer is 2% skimmed milk power+PBS.100uL is added per hole, 1 hour is incubated in 37 DEG C, it is molten to remove antibody
Liquid, PBS solution wash twice.Then to every hole addition dilution buffer, with the ELIAS secondary antibody of 1: 5000 dilution, (HRP is marked
Goat-anti rabbit and the goat-anti cavy of HR marks) each 100ul, 37 DEG C of insulations remove enzyme standard liquid after 0.5 hour, and PBS solution washes twice.
Then 100ul DAB nitrite ions are added into every hole, room temperature lucifuge adds 2M H after acting on 10 minutes2SO40.05mL terminate liquids terminate
Reaction, and determine OD with enzyme mark colour comparatour450Value, and calculating antibody potency, antibody titer value are as shown in table 1.
The animal antibody titer value of table 1
| Rabbit-anti -1 | Rabbit-anti -2 | Cavy anti-1 | Cavy anti-2 | |
| Potency | 1×107 | 1×107 | 1×107 | 1×106 |
As shown in Table 1, the antigen purification mode of the inventive method and immune step can obtain the antibody of high-titer.
The double antibody sandwich method of embodiment 3 determines Hansenula yeast host protein content
Coating：The anti-Hansenula yeast host cell antibody of cavy is diluted to coating buffer (0.05MNaHCO3, pH9.6)
10ug/mL, 100ul/ hole, 4 DEG C of coatings are overnight.Coating buffer is removed, is washed twice with PBS solution.0.3mL confining liquids are added per hole
(5% skimmed milk power+PBS) is incubated 2 hours in 37 DEG C.Coating buffer is removed, PBS solution washes twice.
Sample-adding：By the Hansenula yeast host cell holoprotein sample of above-mentioned acquisition be diluted to 250ng/mL, 125ng/mL,
62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.9ng/mL, dilution buffer are containing 2% skimmed milk power
PBS, each hole adds 100ul dilute samples, and single plus dilution buffer hole is as negative control.1 hour is incubated in 37 DEG C, is removed
Antibody-solutions are removed, PBS solution washes twice.
Colorimetric and result calculate：Then to every hole addition dilution buffer, with the ELIAS secondary antibody of 1: 5000 dilution, (HRP is marked
The goat anti-rabbit igg of note) 100ul, 37 DEG C of insulations remove enzyme standard liquid after 0.5 hour, and PBS solution washes twice.Then into every hole
100ul DAB nitrite ions are added, room temperature lucifuge adds 2M H2SO40.05mL terminate liquid terminating reactions after acting on 10 minutes, and uses enzyme
Mark colour comparatour measure OD450 values.Standard curve is drawn with linear fitting, as shown in Figure 1.As shown in Figure 1, standard curve R2 is
0.998, the sensitivity that Hansenula yeast host protein is detected using this method reaches 4ng/mL.
The prior art control experiment of embodiment 4
As described in above-mentioned embodiment 1, one exempts to six to exempt from all for the preparation of Hansenula yeast host cell holoprotein and immune programme for children
Using Hansenula yeast host cell holoprotein, antibody purification and double sandwich method determination steps are as described in above-mentioned embodiment 3.With linear
Fitting process draws standard curve, as shown in figure 1, standard curve R2 is 0.997, the sensitivity of detection Hansenula yeast host protein is
15.6ng/mL.Testing result of the present invention is shown in Fig. 1 with original technology comparing result, and testing result sensitivity of the invention is higher, line
Property is more preferably.
The host cell residues detection of the Hansenula yeast of embodiment 5 recombination expression hepatitis b surface antigen protein (HBsAg) should
With
1. the expression of recombinant HBsAg
The DNA of HBsAg albumen is encoded with reference to GenBank：AF479684 sequence, codon uses to be used in Hansenula yeast
The higher codon of frequency optimizes, full genome synthesis.
Using the gene and pMOX plasmids of the mixing above-mentioned synthesis of ferment treatment of BstBI enzymes and KpnI enzymes, the fragment after digestion
Connected by T4DNA ligases, the recombinant plasmid transformed bacillus coli DH 5 bacterial strain of acquisition, 37 DEG C are inverted culture overnight.Picking part
Clone's extracting plasmid, is identified with BstBI and KpnI digestions, obtains HBsAg expressed by Hansenula yeast plasmids.
Hansenula yeast bacterium source is in China General Microbiological culture presevation administrative center, CGMCC numberings 2,2497.Will weight
Group HBsAg expressed by Hansenula yeast plasmid ScaI linearization for enzyme restriction, electricity turn Hansenula yeast.Bacterium solution coating YPD flat boards after electricity turns
37 DEG C of (2000g/ml Zeocin) inversion culture overnight 2 days, preferable one plant of picking growing state is expressed as recombinant HBsAg
Bacterial strain.
By recombinant HBsAg expression bacterial strain access 500ml seed culture mediums (YPD culture mediums), 37 DEG C of overnight incubations, by 1: 15
Inoculation fermentation tank.
Using BIOENGINEERINGRALF Basic fermentation tanks, fermentation uses basal salt media BSM.It is wet to detect thalline
When increasing to 100~200g/L again, methanol induction is initially added into, induces 48 hours fermentation ends.
Separation of solid and liquid is carried out to zymotic fluid using refrigerated centrifuge, centrifugal rotational speed 8000rpm, centrifuged 10 minutes.Centrifugation knot
Supernatant is abandoned after beam, collects thalline.
Thalline is with after high-pressure homogenization crusher machine, by serial micro-filtration, ultrafiltration, hydrophobic chromatography, anion chromatography, molecular sieve layer
Analysis, obtain the HBsAg albumen of purifying.
2. the detection of Hansenula yeast host protein residual quantity
Coating：The anti-Hansenula yeast host cell antibody of cavy is diluted to coating buffer (0.05MNaHCO3, pH9.6)
10ug/ml, 100ul/ hole, 4 DEG C of coatings are overnight.Coating buffer is removed, is washed twice with PBS solution.0.3ml confining liquids are added per hole
(5% skimmed milk power+PBS) is incubated 2 hours in 37 DEG C.Coating buffer is removed, PBS solution washes twice.
Sample-adding：It is positive reference product (being used as standard curve) by Hansenula yeast host cell holoprotein (antigen 1) sample, it is dilute
Release to 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.9ng/mL.Restructuring
HBsAg albumen is diluted to 10ug/mL sample-addings, multiple holes measure.Dilution buffer is the PBS containing 2% skimmed milk power, and each hole adds
100ul dilute samples, single plus dilution buffer hole are incubated 1 hour, PBS solution washes twice as negative control in 37 DEG C.
1 hour is incubated in 37 DEG C with the rabbit-anti Hansenula yeast host cell antibody of 1: 5000 dilution per hole addition dilution buffer, PBS
Solution washes twice, and removes antibody-solutions.
Colorimetric and result calculate：Then to every hole addition dilution buffer, with the ELIAS secondary antibody of 1: 5000 dilution, (HRP is marked
The goat anti-rabbit igg of note) 100ul, 37 DEG C of insulations remove enzyme standard liquid after 0.5 hour, and PBS solution washes twice.Then into every hole
100ulDAB nitrite ions are added, after room temperature lucifuge acts on 10 minutes plus 2MH2SO40.05ml terminate liquid terminating reactions, and with enzyme mark
Colour comparatour determines OD450 values.
Same recombinant HBsAg is determined with antibody obtained by above-described embodiment 5 (present invention) and embodiment 4 (control experiment) respectively
Hansenula yeast host cell residual quantity in protein sample, as a result as shown in table 2：Table 2
Finally be necessary described herein be：Above example is served only for further detailed to technical scheme work
Ground explanation, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art is according to the above of the invention
Some the nonessential modifications and adaptations made belong to protection scope of the present invention.
Claims (4)
1. a kind of prepare the low methods for exempting from reinforced antibody of Hansenula yeast HCP, it is characterised in that by routine immunization animal
Antigen purification step is added in method so that animal to Hansenula yeast whole host cell after immune response is produced, reinforcement pair
In the immunoreactivity of part weak immunogenicity host protein, exempt from reinforced HCP antibody so as to be made low, methods described includes
Following steps：
A) several conventional Hansenula yeast strains are chosen and carries out seed culture and induced expression respectively, collect thalline；
B) by the thalline of above-mentioned induced expression respectively, equal weight is respectively taken, is mixed, clasmatosis, separation of solid and liquid, is collected
Bacteria break supernatant, filtration sterilization, Hansenula yeast HCP holoproteins are made；
C) suitable adjuvant immunity animal is added using Hansenula yeast HCP holoproteins, after being immunized for several times, obtains middle antibody；
D) Antibody preparation affinity chromatography glue among using, affinity purification Hansenula yeast HCP holoproteins, it is low to obtain Hansenula yeast HCP
Exempt from albumen；
E) continue immune animal on the basis of step c) using the low albumen of exempting from of Hansenula yeast HCP, after being immunized for several times, it is inferior to obtain the Chinese
Yeast HCP is low to exempt from reinforced antibody,
Wherein, the conventional Hansenula yeast strain described in step a) is wild type, being immunized for several times as three times described in step c)
Immune, described being immunized three times is respectively to be carried out at the 0th, 14,28 day, being immunized for several times to be immunized three times described in step e),
Described be immunized three times is respectively to be carried out at the 42nd, 49,56 day, described conventional Hansenula yeast strain be ATCC14754,
ATCC34438 or ATCC66057.
2. a kind of Hansenula yeast HCP is low to exempt from reinforced antibody, the Hansenula yeast HCP is low, and to exempt from reinforced antibody be Anti-TNF-α
Body, it is made again after antibody purification through animal immune after Hansenula yeast host protein is made with Hansenula yeast bacterial strain, the Chinese is inferior
Yeast HCP is low to exempt from reinforced antibody and is prepared as the method described in claim 1.
3. Hansenula yeast HCP as claimed in claim 2 is low to exempt from reinforced antibody, it is characterised in that in the animal immune also
Including by antigen purification step so that immune animal is strengthened exempting from for weaker after immune response is produced to whole host cells
The immunoreactivity of epidemic focus host protein, the antigen purification step are using animal immune moderate resistance Hansenula yeast host protein
The middle antibody purification Hansenula yeast host cell holoprotein of antibody, obtains that Hansenula yeast host cell is low exempts from albumen.
4. the low application for exempting from reinforced antibody of Hansenula yeast HCP as claimed in claim 2 or claim 3, is used for as active component
Prepare the reagent or kit for the biological products host protein residual quantity that detection is prepared by expressed by Hansenula yeast system.
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| CN109384832B (en) * | 2018-09-29 | 2020-06-09 | 北京民海生物科技有限公司 | Preparation method and application of recombinant vaccine host protein antibody |
| CN115894677A (en) * | 2022-12-01 | 2023-04-04 | 湖州申科生物技术股份有限公司 | Antibody combination for improving HCP detection antibody coverage rate and application thereof |
| CN117192107A (en) * | 2023-09-11 | 2023-12-08 | 福建基诺厚普生物科技有限公司 | Detection method and kit for process-specific host cell protein residues |
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Effective date of registration: 20180411 Address after: 201318 No. 908 lane, zipping Road, Pudong New Area, Shanghai 12-13 Co-patentee after: Chongqing bloomer bio Pharmaceutical Co., Ltd. Patentee after: Shanghai Bovax Biological Science & Technology Co., Ltd. Address before: 201318 306, 307, building 2, 3377 Kang Xin Road, Pudong New Area, Shanghai. Patentee before: Shanghai Bovax Biological Science & Technology Co., Ltd. |